Antimicobial peptides for inhibiting drug-resistant bacteria and uses thereof

ABSTRACT

Disclosed are antimicrobial peptides capable of inhibiting and killing multiple drug-resistant bacteria, including XH-12C, XH-12B and XH-12A. The application further provides uses of the antimicrobial peptides in the preparation of a drug for inhibiting and/or killing fungi, Gram-positive bacteria, Gram-negative bacteria and drug-resistant bacteria and in the manufacture of medical carriers.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Patent ApplicationNo. PCT/CN2017/109799, filed on Nov. 7, 2017, which claims the benefitof priority from Chinese Application No. 201610983926.6, filed on Nov.9, 2016. The contents of the aforementioned applications, including anyintervening amendments thereto, are incorporated herein by reference inits entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (Untitled_ST25.txt;Size: 1,000 bytes; and Date of Creation: Jul. 21, 2019) is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The application relates to antimicrobial peptides, and particularly toan antimicrobial peptide capable of inhibiting and killing bacteria, andmore specifically to an antimicrobial peptide capable of inhibiting andkilling multiple drug-resistant bacteria.

BACKGROUND

The misuse of antibiotics results in a high level of drug resistance andmultidrug resistance (MDR) in pathogenic microorganisms, which hasbecome a worldwide problem. Recently, the “superbugs” with MDR havespread rapidly around the world, seriously threatening human life andhealth. Studies have shown that pathogenic bacteria can obtain MDRthrough some mechanisms such as horizontal gene transfer to resist avariety of antibiotics used clinically. The modification of existingantibiotics and development of new antibiotics are conducted to overcomethe challenges from “superbugs”, in addition to which, alternatives ofantibiotics are expected to be developed to alleviate the internationalhealth security crisis caused by the “superbugs”.

SUMMARY

The present invention provides an antimicrobial peptide capable ofinhibiting and killing multiple drug-resistant bacteria and usesthereof.

An antimicrobial peptide capable of inhibiting and killing multipledrug-resistant bacteria of the application is any one peptide selectedfrom the group consisting of:

XH-12C:  Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Trp-Arg shown as SEQ ID NO. 1; XH-12B: Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile- Phe shown as SEQ ID NO. 2; and XH-12A: Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile  shown as SEQ ID NO. 3.

The application further discloses a use of the above antimicrobialpeptide in the preparation of a drug for inhibiting and/or killing afungus, a Gram-positive bacterium, a Gram-negative bacterium and adrug-resistant bacterium.

In an embodiment, the fungus is Candida albicans; the Gram-positivebacterium is Staphylococcus aureus and Listeria monocytogenes; theGram-negative bacterium is Escherichia coli; and the drug-resistantbacterium is drug-resistant Staphylococcus aureus, drug-resistantAcinetobacter baumannii, drug-resistant Klebsiella pneumoniae,drug-resistant Enterobacter sakazakii, drug-resistant Salmonellagallinarum, drug-resistant Streptococcus agalactiae, drug-resistantEnterococcus faecalis or drug-resistant Riemerella anatipestifer.

The application also provides a use of the antimicrobial peptide in thepreparation of a drug for treating a cancer.

In an embodiment, the cancer comprises cancer cells selected from H460,KB-3-1 or drug-resistant cancer cells MX20, KB-C2 or KB-CV60.

The present invention further provides a use of the antimicrobialpeptide in the manufacture of a medical carrier.

In an embodiment, the drug comprises at least one of XH-12C, XH-12B, andXH-12A. In an embodiment, the drug is mixed with at least onepharmaceutically acceptable carrier or additive.

In an embodiment, the antimicrobial peptide is applied in any one ofhuman drug (for antimicrobial, anticancer and burn treatment),veterinary drug, food additive, feed additive and daily chemicalproduct.

The antimicrobial peptide of the present invention is prepared by a BOCmethod in the existing solid phase peptide synthesis. The BOC methodincludes:

(1) synthesis of resin

Peptide acid Merrifield resin and PAM resin;

Peptide carboxamide MBHA resin;

(2) synthesis of a peptide chain

2.1 coupling of amino acids

2.2 removal of an N-terminal Boc group

removing the N-terminal Boc protective group with TFA before HFcleavage, where the N-terminal Boc group is artificially cleaved bywashing with a solution of TFA and DCM in a ratio of 1:1 at roomtemperature for 15 minutes;

(3) cleavage

3.1 treatment of resin before the cleavage

completely washing and drying the resin before the cleavage; where theresin may be treated as follows in some special cases:

A) cleavage of a dinitrophenyl (DNP) protective group of His

swelling the resin with a minimum volume of DMF; treating the resin with20 mol of thiophenol for 1-2 hours; transferring the resin to a bondingglass funnel; and washing the resin frequently with a HF solution, orbefore treatment with TFMSA, washing the resin with DMF, methanol andcold ethyl ether;

B) deformylation of a Trp-containing polypeptide resin

adding a solution of piperidine and DMF at a volume ratio of 1:10 to around-bottom flask; cooling the solution in an ice bath; introducing thepolypeptide resin (1 g/10 mL) to the solution followed by stirring at 0°C. for 2 hours for reaction; washing the resin twice with DMF (5 timesthe volume of the resin), twice with DCM and twice with MeOH; and dryingthe resin for at least 4 hours under high vacuum before the cleavagewith HF;

3.2 HF cleavage

standard HF-cleaving method (0.2 mmol)

A) adding the polypeptide resin, a polytetrafluoroethylene tube and apurificant mixture to a reaction vessel, where a purificant mixture ofHF, anisole, DMS and p-tolyl mercaptan in a ratio of 10:1:1:0.2 is usedto treat a polypeptide with Cys, while a polypeptide without Cys istreated with a purificant mixture of HF DMS and anisole in a ratio of10:1:1;

B) screwing a cap tightly and cooling the reaction mixture in an icypure methanol for at least 5 minutes;

C) distilling 10 mL of HF to a bottle following the instructions of themanufacture, since it may take more than 2 hours for cleavage of an Arg(Tos)-containing polypeptide;

D) at the end of the reaction, evaporating HF and DMS by nitrogen steam;

E) absorbing the cleaved polypeptide from the resin with TFA;

F) collecting the resin by filtration under vacuum; washing the resintwice with TFA followed by filtration to obtain a filtrate; and addingcold ethyl ether (8-10 times the volume of the filtrate) to thefiltrate; where in some cases, there is a need to evaporate most of theTFA to obtain a precipitate of a crude product;

3.3 post-processing of the cleavage

A) precipitation

collecting the precipitated peptide by filtration with a hard filterpaper in a Hirsch funnel under vacuum; washing the precipitated peptidewith cold ethyl ether; and dissolving the precipitated peptide in anappropriate buffer solution followed by lyophilization;

B) centrifugation

adding a small volume of tert-butyl methyl ether to the lyophilizedproduct followed by grinding until a suspended substance is produced;transferring the suspended substance to a clean centrifuge tube followedby hermetic centrifugation, where an automatic centrifuge is necessary;pouring the ether carefully out of the tube and washing the precipitaterepeatedly with the ether; and dissolving the precipitate in anappropriate buffer solution followed by lyophilization;

C) after the water-soluble peptide is precipitated, adding water to theprecipitate to produce a mixture; and transferring the mixture to aseparatory funnel to which a small amount of ethanol may be added topromote dissolution;

D) shaking the sealed funnel thoroughly to disperse the mixture followedby standing for separation; and collecting the lower-layer liquid(water);

E) adding a small amount of water to the funnel; repeating the processof shaking-standing-separation three times; collecting the lower-layerliquid to a clean flask and removing the upper-layer liquid; andtransferring the lower-layer liquid to the funnel;

F) adding a small amount of a freshly prepared diethyl ether; repeatingthe process of shaking-standing-separation two to three times, where theether layer is removed each time and the water layer is recycled to thefunnel; and after that, collecting the water layer to a clean flaskfollowed by lyophilization to produce the antimicrobial peptide.

A use of the antimicrobial peptide of the invention in the preparationof, for example a drug for treating the infection caused byGram-positive bacteria such as Staphylococcus aureus and Listeriamonocytogenes or Gram-negative bacteria such as Escherichia coli,particularly in the preparation of a human drug or a veterinary drug forkilling and inhibiting Staphylococcus aureus, Listeria monocytogenesand/or Escherichia coli.

The drug may comprise one or more of the antimicrobial peptidesdisclosed herein.

The drug may further comprise one or more pharmaceutically acceptablecarriers or additives, such as an active enzyme for promoting enzymatichydrolysis, or an activator for enhancing dispersion.

The dosage and use conditions of the antimicrobial peptide of theinvention in the above use methods can be determined by the methodsknown in the art.

It has been experimentally demonstrated that the antimicrobial peptideof the present invention plays a significant role in inhibiting andkilling Gram-positive bacteria, particularly Staphylococcus aureus andListeria monocytogenes, Gram-negative bacteria, particularly Escherichiacoli, or fungi, particularly Candida albicans. In addition, theapplication also has a significant effect on multiple drug-resistantbacteria, ordinary cancer cells and drug-resistant cancer cells.

In summary, the antimicrobial peptide of the invention has abroad-spectrum antimicrobial activity, a low hemolytic activity and ananti-cancer activity, and particularly provides a desirable therapeuticeffect on drug-resistant bacteria and drug-resistant cancer cells.Therefore, the present invention is promising in the applications ofmedicines, food and daily chemical products.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the invention are further described in detail belowwith reference to the accompanying drawings.

FIGS. 1A-C show the effect of the antimicrobial peptide XH-12A onkilling Staphylococcus aureus (A), Listeria monocytogenes (B), andEscherichia coli (C).

FIGS. 2A-C show the effect of the antimicrobial peptide XH-12B onkilling Staphylococcus aureus (A), Listeria monocytogenes (B), andEscherichia coli (C).

FIGS. 3A-C show the effect of the antimicrobial peptide XH-12C onkilling Staphylococcus aureus (A), Listeria monocytogenes (B), andEscherichia coli (C).

DETAILED DESCRIPTION OF EMBODIMENTS

The invention will be further illustrated below with reference to theembodiments, but are not limited thereto.

Example 1 Evaluation of Minimum Inhibitory Concentration (MIC) UsingMicro-Broth Dilution Method

(A) Preparation of Antimicrobial Drug and Medium

The antimicrobial drug and the medium were prepared according to aconstant broth dilution method.

Concentration range of the antimicrobial drug for susceptibility testincludes boundary points of the resistant, intermediate and susceptiblevalues according to the NCCLS Antimicrobial Susceptibility TestOperating Standard.

A Mueller-Hinton (MH) broth at pH of 7.2-7.4 recommended by NCCLS wasused herein as the medium in which aerobic bacteria and facultativeanaerobic bacteria grew well.

Preparation of Inoculum

3-5 colonies to be examined of similar morphology were picked up by aninoculating loop, inoculated in 4-5 mL of casein hydrolysate (MH) brothand cultured at 35° C. for 2-6 hours. After the enrichment, thebacterial suspension at the logarithmic phase was adjusted with normalsaline or MH broth to a concentration of 0.5 McFarland standard, i.e.,about 1×10⁸-2×10⁸ CFU/mL.

B) Preparation of MIC Plate

Under sterile conditions, different concentrations of the antimicrobialdrug solutions obtained by multiple dilution were respectively added toa sterilized 96-well polystyrene plate, where the 1^(st) to the 11^(th)wells were added with the drug solution at 10 μL per well and the12^(th) well free of drug solution was used as the growth control. Thenthe 96-well plate was lyophilized, sealed and stored at −20° C. for use.

C) Preparation of Inoculum

A bacterial suspension prepared by a growth method or a direct bacterialsuspension method and having a concentration equivalent to 0.5 McFarlandstandard was diluted with MH broth in a ratio of 1:1,000 and added tothe 96-well plate at 100 μL per well. The plate was sealed and incubatedat 35° C. in an ordinary air incubator for 16-20 hours for assessment,where drug concentrations of the 1^(st) to the 11^(th) wells were 128,64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/mL, respectively.

D) Results

The results were assessed as follows. The minimum concentration at whichthe drug completely inhibits the bacterial growth in the wells was theminimum inhibitory concentration (MIC). The test was useful only whenthe bacteria grew significantly in the positive control well (i.e., noantibiotics). When a single drift occurred in the micro-broth dilutionmethod, the highest concentration at which the drug inhibited thebacterial growth was recorded.

The results were shown in Table 1.

TABLE 1 Staphylococcus Listeria Escherichia Peptides aureusmonocytogenes coli XH-12A  4 μg/mL 4 μg/mL 32 μg/mL XH-12B 16 μg/mL 16μg/mL  64 μg/mL XH-12C 16 μg/mL 8 μg/mL 32 μg/mL

Staphylococcus aureus, Listeria monocytogenes and Escherichia coli usedin this test were ATCC25922, ATCC19115 and ATCC29213, respectively.

Example 2 Minimum Bactericidal Concentration (MBC) Test

The minimum bactericidal concentration referred to a minimumconcentration required for killing 99.9% (reduced by 3 orders ofmagnitude) of the test microorganisms.

The determination of the minimum bactericidal concentration using adoubling dilution method was performed by halving the drugconcentrations in order. For example, when the minimum bactericidalconcentration (MBC) was determined in a certain row/column of a 96-wellplate, each well was added with 10 μL of different concentrations of thedrug solution to provide the first well with a final drug concentrationof 128 g/mL, the second well with a final drug concentration of 64 μg/mLand the third well with a final drug concentration of 32 μg/mL, and soon. A lowest drug concentration at which no viable cells were observedwas the MBC. The results were shown in FIGS. 1A-1C, 2A-2C and 3A-3C.

In FIGS. 1A-1C, 2A-2C and 3A-3C, HR referred to hour; concentration waspresented by μg/mL; and CFU reduction % referred to percentage forcolony reduction.

Staphylococcus aureus, Listeria monocytogenes and Escherichia coli usedin this test were ATCC25922, ATCC19115 and ATCC29213, respectively.

Example 3 Determination of Inhibitory Activity and Minimum InhibitoryConcentration (MIC) Against Multidrug-Resistant Bacteria

A doubling dilution method was used to determine the minimum inhibitoryconcentration. The test strains were multidrug-resistant Acinetobacterbaumannii, Klebsiella pneumonia, Staphylococcus aureus, Enterobactersakazakii, Salmonella gallinarum, Streptococcus agalactiae, Enterococcusfaecalis and Riemerella anatipestifer. Each of the strains wasinoculated to an MH solid medium and incubated invertedly at 37° C. inan incubator. After the colonies appeared, the single colony wastransferred to an MH liquid medium by an inoculating loop and incubatedat 37° C. under shaking in an incubator to the logarithmic growth phase.Then OD₆₀₀ of the bacterial liquid was measured by an ultravioletspectrophotometer, and the concentration of the bacterial liquid wascalculated according to 1 OD₆₀₀≈1×10⁹ CFU/mL. After that, the bacterialliquid was diluted with the MH medium to 2×10⁵ CFU/mL. 90 μL of thediluted bacterial liquid and 10 μL of a peptide solution were added andmixed uniformly in each well of a 96-well plate, incubated at 37° C. for18 hours and measured by a microplate reader for OD₆₀₀. The minimumconcentration of a sample to inhibit the bacterial growth was theminimum inhibitory concentration (MIC) of the sample. The experiment wasrepeated 3 times and the results were averaged and shown in Tables 2-3.

TABLE 2 Minimum inhibitory concentration of antimicrobial peptidesagainst multidrug-resistant bacteria in human multidrag- multidrug-multidrug- resistant resistant resistant Staphylococcus AcinetobacterKlebsiella aureus baumannii pneumoniae XH-12C 4 μg/mL 4 μg/mL 4 μg/mLXH-12B 4 μg/mL 8 μg/mL 4 μg/mL XH-12A 4 μg/mL 8 μg/mL 8 μg/mL

TABLE 3 Minimum inhibitory concentration of antimicrobial peptidesagainst multidrug-resistant bacteria in animals Enterobacter SalmonellaStreptococcus Enterococcus Riemerella sakazakii gallinarum agalactiaefaecalis anatipestifer XH-12C 300 μg/mL 300 μg/mL 50 μg/mL 200 μg/mL 300 μg/mL XH-12B 300 μg/mL 300 μg/mL 50 μg/mL 50 μg/mL 300 μg/mL XH-12A250 μg/mL 300 μg/mL 50 μg/mL 50 μg/mL 300 μg/mL

The experimental results showed that the above antimicrobial peptideshad inhibitory activities against multidrug-resistant bacteria in bothhuman and animals.

Example 4 Determination of Inhibitory Activity and Minimum InhibitoryConcentration (MIC) Against Fungi

According to the M27-A protocol of NCCLS, the antimicrobial peptide wasdiluted by doubling dilution and 100 μL of the diluted peptide was addedto a 96-well plate followed by adding of 100 μL of a suspension at0.5-2.5×10³ Candida albicans/mL and cultured at 37° C. for 48 hours. Theminimum peptide concentration at which no bacterial growth was observedwas the MIC of the antimicrobial peptide against Candida albicans. Theexperiment was repeated 3 times and the results were averaged and shownin Table 4.

TABLE 4 Minimum inhibitory concentration of antimicrobial peptidesagainst fungi Candida albicans XH-12C 4 μM XH-12B 146 μM XH-12A 20 μM

The experimental results showed that the above antimicrobial peptides,especially the antimicrobial peptides XH-12C and XH-12A, had inhibitoryactivities against Candida albicans.

Example 5 Determination of Inhibitory Activities and Half MaximalInhibitory Concentration (IC₅₀) Against Drug-Resistant Cancer Cells

The toxicity of antimicrobial peptides to cancer cells was analyzed byMTT assay.

The cells were first digested with trypsin and suspended in a culturedish. The cell suspension was seeded in a 96-well plate at 180 μL perwell to obtain a final concentration of 5×10³ cells/well, and incubatedfor 24 hours. Different concentrations of peptide in each 20 μL wereadded, and topotecan, paclitaxel, vincristine and doxorubicin were usedas positive controls. The plate was continuously incubated for 68 hoursand added with 4 mg/mL of MTT reagent followed by another incubation for4 hours. The supernatant was discarded and the crystallate was dissolvedin 100 μL of DMSO for measurement of cell viability at 570 nm. The IC₅₀value was calculated according to the survival curve. The experiment wasrepeated 3 times and the results were averaged and shown in Table 5.

TABLE 5 Minimum inhibitory concentration of antimicrobial peptidesagainst cancer cells and drug-resistant cancer cells H460 MX20 KB-3-1KB-C2 KB-CV60 XH-12C 6 μM 6 μM 6 μM 3 μM 6 μM XH-12B 8 μM 7 μM 5 μM 4 μM7 μM XH-12A 75 μM  49 μM  20 μM  22 μM  25 μM 

In Table 5, MX20 was a drug-resistant H460 cell strain overexpressingBCRP protein and induced by Mitoxantrone; KB-C2 was a drug-resistantKB-3-L cell strain overexpressing P-gp protein and induced byColchicine; and KB-CV60 was a drug-resistant KB-3-1 cell strainoverexpressing MRP1 protein and co-induced by Cepharanthine andVincritine.

The experimental results showed that the above antimicrobial peptideshad inhibitory activities against both cancer cells and drug-resistantcancer cells.

Example 6 Determination of Hemolytic Activity

1) Fresh human red blood cells were placed in a centrifuge tubecontaining heparin anticoagulant and centrifuged at 1,200 rpm for 15minutes. The supernatant was discarded and the cells were washed severaltimes with normal saline. The resulting red blood cells were preparedinto a 2% (v/v) suspension with PBS. 100 μL of the cell suspension and100 μL of an antimicrobial peptide solution (at a concentration of16-3,200 μg/mL) were placed in a 96-well plate, incubated at 37° C. for2 hours, centrifuged at 1,200 rpm for 10 minutes and measured at 620 nmby a microplate reader for the absorbance D. The hemolysis rate wascalculated according to the equation: Hemolysis rate(%)=(D_(test)−D_(negative))/(D_(positive)−D_(negative))×100%. Theexperiment was repeated three times and the results were averaged. Withreference to criteria for hemolysis test in the test methods of infusionequipment, transfusion equipment and injector for medical use, ahemolysis rate less than 5% indicated a negative reaction, i.e., theabsence of hemolysis; and a hemolysis rate greater than 5% indicated apositive reaction, i.e., the occurrence of hemolysis. The results wereshown in Table 6.

TABLE 6 Hemolytic activity of antimicrobial peptides for human red bloodcells Concentration for hemolysis rate less than 5% XH-12C 64 μg/mLXH-12B 80 μg/mL XH-12A 320 μg/mL 

The experimental results indicated no adverse effect of the aboveantimicrobial peptides on human red blood cells.

2) 6 mL of blood was collected from a heart of a healthy rabbit,immediately mixed with Alsever's solution in a ratio of 1:1, placed in acentrifuge tube and centrifuged at 1,200 rpm for 15 minutes. Thesupernatant was discarded and the cells were washed several times withnormal saline. The resulting red blood cells were prepared into a 2%(v/v) suspension with PBS. 100 μL of the cell suspension and 100 μL ofan antimicrobial peptide solution (at a concentration of 16-3200 μg/mL)were placed in a 96-well plate, incubated at 37° C. for 2 hours,centrifuged at 1,200 rpm for 10 minutes and measured at 540 nm by amicroplate reader for the absorbance D. The hemolysis rate wascalculated according to the equation: Hemolysis rate(%)=(D_(test)−D_(negative))/(D_(positive)−D_(negative))×100%. Theexperiment was repeated three times and the results were averaged. Withreference to criteria for hemolysis test in the test methods of infusionequipment, transfusion equipment and injector for medical use, ahemolysis rate less than 5% indicated a negative reaction, i.e., theabsence of hemolysis; and a hemolysis rate greater than 5% indicated apositive reaction, i.e., the occurrence of hemolysis. The results wereshown in Table 7.

TABLE 7 Hemolytic activity of antimicrobial peptides for rabbit redblood cells Concentration for hemolysis rate less than 5% XH-12C  80μg/mL XH-12B 160 μg/mL XH-12A 320 μg/mL

The experimental results indicated no adverse effect of the aboveantimicrobial peptides on mammalian red blood cells.

Finally, it should also be noted that listed above are merely someembodiments of the invention. Obviously, the invention is not limited tothe above embodiments and many variations of the embodiments can bemade. All variations that can be directly derived or conceived by thoseskilled in the art from the disclosure of the invention should stillfall within the scope of the invention.

We claim:
 1. An antimicrobial peptide capable of inhibiting and killingmultiple drug-resistant microorganisms, wherein the antimicrobialpeptide is any one peptide selected from the group consisting of:XH-12C:  Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile-Trp-Arg shown as SEQ ID NO. 1; XH-12B: Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile- Phe shown as SEQ ID NO. 2; and XH-12A: Phe-Phe-Arg-Lys-Val-Leu-Lys-Leu-Ile-Arg-Lys-Ile  shown as SEQ ID NO. 3.